RESUMEN
BACKGROUND: Realistic reconstruction of the in vivo human atherosclerotic environment requires the coculture of different cell types arranged in atherosclerotic vessel-like structures with exposure to flow and circulating cells, presenting challenges for disease modeling. This study aimed to develop a 3-dimensional tubular microfluidic model with quadruple coculture of human aortic smooth muscle cells, human umbilical cord vein endothelial cells, and foam cells to recreate a complex human atherosclerotic vessel in vitro to study the effects of flow and circulating immune cells. METHODS: We developed a coculture protocol utilizing BFP (blue fluorescent protein)-labeled human aortic smooth muscle cells, GFP (green fluorescent protein)-labeled human umbilical cord vein endothelial cells, and THP-1 macrophage-derived, Dil-labeled oxidized LDL (low-density lipoprotein) foam cells within a fibrinogen/collagen I-based 3-dimensional ECM (extracellular matrix). Perfusion experiments were conducted for 24 hours on both atherosclerotic vessels and healthy vessels (BFP-labeled human aortic smooth muscle cells and GFP-labeled human umbilical cord vein endothelial cells without foam cells). Additionally, perfusion with circulating THP-1 monocytes was performed to observe cell extravasation and recruitment. RESULTS: The resulting vessels displayed early lesion morphology, with a layered composition including an endothelium and media, and foam cells accumulating in the subendothelial space. The layered wall composition of both atherosclerotic and healthy vessels remained stable under perfusion. Circulating THP-1 monocytes demonstrated cell extravasation into the atherosclerotic vessel wall and recruitment to the foam cell core. The qPCR analysis indicated increased expression of atherosclerosis markers in the atherosclerotic vessels and adaptation of vascular smooth muscle cell migration in response to flow and the plaque microenvironment, compared with control vessels. CONCLUSIONS: The human 3-dimensional atherosclerosis model demonstrated stability under perfusion and allowed for the observation of immune cell behavior, providing a valuable tool for the atherosclerosis research field.
RESUMEN
Living myocardial slices (LMS) are beating sections of intact human myocardium that maintain 3D microarchitecture and multicellularity, thereby overcoming most limitations of conventional myocardial cell cultures. We introduce a novel method to produce LMS from human atria and apply pacing modalities to bridge the gap between in-vitro and in-vivo atrial arrhythmia studies. Human atrial biopsies from 15 patients undergoing cardiac surgery were dissected to tissue blocks of ~ 1 cm2 and cut to 300 µm thin LMS with a precision-cutting vibratome. LMS were placed in a biomimetic cultivation chamber, filled with standard cell culture medium, under diastolic preload (1 mN) and continuous electrical stimulation (1000 ms cycle length (CL)), resulting in 68 beating LMS. Atrial LMS refractory period was determined at 192 ± 26 ms. Fixed rate pacing with a CL of 333 ms was applied as atrial tachyarrhythmia (AT) model. This novel state-of-the-art platform for AT research can be used to investigate arrhythmia mechanisms and test novel therapies.